Instrument: Illumina HiSeq 2000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: Excess formaldehyde was quenched by addition of glycine (0.625M). Cells were washed with cold PBS and spun down (3,000 rpm for 10 minutes at 4°C; Kubota 3500). The pellet was re-suspended in FA lysis buffer (0.25% Triton X-100, 10 mM EDTA, 10 mM Tris HCl [pH 8.0], 100 mM NaCl, Roche 1X cOmplete protease inhibitor) and incubated for 15 minutes. Nuclei were collected (3,000 rpm for 10 minutes at 4°C; Kubota 3500) and re-suspended in 300 μl SDS lysis buffer (1% SDS, 1% Triton X 100, 2 mM EDTA, 50 mM Hepes-KOH [pH 7.5], 0.1% Na dodecyl sulfate, Roche 1X cOmplete protease inhibitor). Nuclei were lysed for 15 minutes, after which sonication was used to fragment chromatin to an average size of 200–500 bp (Bioruptor Next gen, Diagenode). Cellular debris was removed by centrifugation at 15,000 rpm at 4°C (Kubota 3500). Next, 300 μl of nuclear lysate was diluted 1:10 with a dilution buffer (1% Triton X 100, 2 mM EDTA, 50 mM Hepes-KOH [pH 7.5], 0.1% Na dodecyl sulfate, Roche 1X cOmplete protease inhibitor) and protein-DNA complexes were immuno-precipitated using 3 μg of H3K27acetyl antibody (AB4729, Lot number 730178, Abcam) coupled to 50μl protein G Dynal beads (Invitrogen) overnight. The beads were washed and protein-DNA complexes were eluted with 150 μl of elution buffer (1% SDS, 10 mM EDTA, 50 mM Tris.HCl [pH 8]), followed by protease treatment and de-crosslinking at 65°C overnight. After phenol/chloroform extraction, DNA was purified by ethanol precipitation. 80% of ChIP material was used for library preparation using the Biooscientific library preparation kit with the following modification. The provided adapters were diluted down 40-fold in order to prevent adapter dimer formation that would affect enrichment. Libraries were then enriched for 15 cycles with PFX polymerase (Invitrogen) followed by gel size selection (300-500bp). The ChIP libraries were then sequenced on the Illumina Hiseq 2000 platform in three batches